1. Field of the Invention
The present invention relates to antibodies specific for epitopes of human glucocorticoid receptor in which a serine residue is phosphorylated. The present invention also relates to a method for determining the presence of activated glucocorticoid receptors and to a method for screening a glucocorticoid agonist.
2. Description of the Related Art
The glucocorticoid receptor (GR) is a phosphoprotein that regulates a wide range of metabolic and developmental processes by controlling the expression of target genes in a hormone-dependent and cell-specific manner (Reichardt et al., 2000 and Yamamoto et al., 1985). GR is structurally similar to other members of the nuclear receptor superfamily, in that separate receptor activities such as DNA and ligand binding are localized to distinct regions of the protein (Weinberger et al., 1985 and Hollenberg et al., 1985). GR contains a constitutive activation function, AF-1, in the N-terminus, and a ligand-dependent AF-2 at the C-terminus (Godowski et al., 1987). In absence of ligand, the hsp90-based chaperone complex represses GR regulatory activities (Pratt et al., 1997). Hormone binding relieves this repression and results in a conformational change in the receptor, which, in turn, promotes GR DNA binding, association with additional transcriptional regulatory cofactors and regulation of target genes (Jenkins et al., 2001).
Although ligand binding is essential for the activation of GR, the receptor is also subject to post-translational modification through phosphorylation (Bodwell et al., 1998). GR is phosphorylated in the absence of hormone, with additional phosphorylation occurring in conjunction with agonist, but not antagonist binding. Orti et al. showed that GR is hyperphosphorylated after it has become activated and acquires the ability to bind to DNA (Orti et al., 1993). It has been suggested that hormone-dependent phosphorylation of GR may determine target promoter specificity, cofactor interaction, strength and duration of receptor signaling, and receptor stability (Garabedian et al., 1998).
Bodwell et al. have identified seven phosphorylation sites in the mouse GR (mGR) over-expressed in Chinese hamster ovary cells by direct sequencing of phosphorylated peptides (Bodwell et al., 1991). All seven phosphorylated residues are clustered in the N-terminal region of the receptor. Through peptide mapping and mutagenesis studies, the laboratory of the present inventors has identified four predominant sites of phosphorylation on rat GR (rGR) expressed in mammalian cells and in yeast, that coincide with a subset of sites identified in mGR (Pocuca et al., 1998 and Krstic et al., 1997). Among them, two sites S224 and S232 corresponding to S203 and S211 in human GR were phosphorylated to a greater extent in the presence of hormone. The laboratory of the present inventors has also identified the cyclin-dependent kinases (Cdks) as potential kinases that modify S224 and S232 in vitro. Mutations in the Cdk catalytic subunit, p34CDC28 or regulatory cyclin subunits reduced receptor-dependent transcriptional activation in a reconstituted GR signaling system in yeast, indicating that Cdk function is necessary for full receptor-mediated transcriptional enhancement (Krstic et al., 1997).
Previous studies by Mason and Housley suggest that single or multiple phosphorylation site mutations in mouse GR (mGR) had little effect on receptor transcriptional activation, subcellular localization or activity in response to cAMP treatment (Mason et al., 1993). Similar results were obtained by Almlof et al. when phosphorylation site mutants in human GR where analyzed in yeast (Almlof et al., 1995). Webster et al. reported that single or multiple phosphorylation site mutations had little effect on mGR expression, nuclear translocation and transcriptional activation from a complex MMTV promoter (Webster et al., 1997). Importantly, they also showed that the phosphorylation status of mGR had a substantial effect on transcriptional activation from a GR-responsive reporter containing a minimal E1b-promoter, suggesting that the effect of GR phosphorylation on transcriptional activation appears promoter-specific. However, the mechanism by which phosphorylation affects GR transcriptional regulation remains enigmatic.
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